Glycosylation is one of most prevalent posttranslational modifications (PTMs) of proteins and plays an important role in many biological processes. For example, the posttranslational modification of proteins by glycosylation can affect protein folding and stability, modify the intrinsic activity of proteins, and modulate their interactions with other biomolecules. See, e.g., Varki, A. (1993) Glycobiology 3:97-130; Dwek (1996) Chem. Rev. 96:683-720; and Sears and Wong (1998) Cell Mol. Life Sci. 54:223-252. Natural glycoproteins are often present as a population of many different glycoforms, which makes analysis of glycan structure and the study of glycosylation effects on protein structure and function difficult. Therefore, methods for the synthesis of natural and unnatural homogeneously glycosylated proteins would be useful tools, e.g., for the systematic understanding of glycan function, and for the development of improved glycoprotein therapeutics.
Considerable effort has focused on the methods for generation of glycoproteins, including chemical and enzymatic synthetic approaches, in vitro translation, and pathway engineering. One previously known approach for making proteins having desired glycosylation patterns makes use of glycosidases to convert a heterogeneous natural glycoprotein to a simple homogenous core, onto which saccharides can then be grafted sequentially with glycosyltransferases. See, e.g., Witte, K., et al., (1997) J. Am. Chem. Soc. 119:2114-2118. A limitation of this approach is that the primary glycosylation sites are predetermined by the cell line in which the protein is expressed. Alternatively, a glycopeptide containing the desired glycan structure can be synthesized by solid phase peptide synthesis. This glycopeptide can be coupled to other peptides or recombinant protein fragments to afford a larger glycoprotein by native chemical ligation (see, e.g., Shin, Y., et al., (1999) J. Am. Chem. Soc. 121:11684-11689) expressed protein ligation, (see, e.g., Tolbert, T. J. and Wong, C.-H. (2000) J. Am. Chem. Soc. 122:5421-5428), or with engineered proteases. See, e.g., Witte, K., et al., (1998) J. Am. Chem. Soc. 120:1979-1989. Both native chemical ligation and expressed protein ligation are most effective with small proteins, and necessitate a cysteine residue at the N-terminus of the glycopeptide. When a protease is used to ligate peptides together, the ligation site is preferably placed far away from the glycosylation site for good coupling yields. See, e.g., Witte, K., et al., (1998) J. Am. Chem. Soc. 120:1979-1989. A third approach is to modify proteins with saccharides directly using chemical methods. Good selectivity can be achieved with haloacetamide saccharide derivatives, which are coupled to the thiol group of cysteine, (see, e.g., Davis, N. J. and, Flitsch, S. L. (1991) Tetrahedron Lett. 32:6793-6796; and, Macmillan, D.; et al., (2002) Org Lett 4:1467-1470), but this method can become problematic with proteins that have more than one cysteine residue.
Accordingly, a need exists for improved methods for making glycoproteins having a desired glycosylation pattern. The invention fulfills this and other needs, as will be apparent upon review of the following disclosure.